The storage of genetic information for all living systems is derived from the organizational string of just two base pairs (A:T and G:C). The simplicity of a two pair code is fascinating yet begs the question; are more than two possible? At least chemically, it has been shown that additional base pairs are possible.
The rule-based molecular recognition displayed by DNA makes it ideal throughout biotechnology, where molecules binding to molecules are needed for such applications as localizing DNA (see, e.g., Pease, A. C., et al., Proc. Natl. Acad. Sci. USA 91:5022-6 (1994)), assembling nanostructures (see, e.g., Collins, M. L., et al., Nucleic Acids Research 25:2979-2984 (1997)), building antibody-like molecules called “aptamers” (see, e.g., Prudent, J. R., et al., Science 264:1924-1927 (1994); and Hermann, T. and Patel, D. J., Science 287:820-825 (2000)) and performing tasks by which information in a molecular structure is recognized. Expanding DNA chemistry to include additional base pairs would enhance the capabilities of this powerful molecular recognition system. This in turn has lead chemists to develop new ways of increasing the number of DNA building blocks. The first experimental data suggesting that new base pairs could be used in replication, transcription and translation was demonstrated by Benner and colleagues using shuffled hydrogen bonding schemes, e.g., Piccirilli, J. A., et al., Nature 343:33-37 (1990) and Switzer, C. Y., et al., J. Am. Chem Soc. 111:8322-8323 (1989). More recently, Romesberg and colleagues used the idea of hydrophobic interactions to create base pairs that did not rely on hydrogen bonding as disclosed in McMinn, D. L., et al., J. Am. Chem Soc. 121:11585-11586 (1999) and Tae, E. L., et al., J. Am. Chem. Soc. 123:7439-7440 (2001). With a specific mixture of two polymerases, the authors demonstrated incorporation followed by extension. Going one step further, Yokoyama and colleagues combined the concepts of shuffled hydrogen bonds and van der Waals interactions to develop a base pair that could be polymerized into RNA transcripts site specifically opposite the non-natural counterpart, e.g., Ohtsuki, T., et al., Proc. Natl. Acad. Sci. USA 98:4922-4925 (2001) and Mitsui, T., et al., J. Am. Chem. Soc. 125:5298-5307 (2003). More recently, it has been demonstrated that consecutive non-natural bases could be incorporated specifically opposite their non-natural counterparts and that other replication dependent enzymes can efficiently recognize a third base pair as disclosed in Moser, M. J., and Prudent, J. R., Nucleic Acids Research 31:5048-53 (2003). Yet the ability to place a third base pair into a commonly used replication system such as the polymerase chain reaction (PCR) has not been demonstrated.